Mammalian soluble adenylyl cyclase (sAC) is structurally, biochemically, and physiologically distinct from G protein coupled transmembrane adenylyl cyclases. sAC activity is not regulated by the known modulators of transmembrane adenylyl cyclases activity, such as G proteins and forskolin, but it is directly stimulated by the bicarbonate- ion. Multiple physiological processes (i.e., breathing, blood flow, cerebrospinal fluid and aqueous humor formation, spermatocyte development) are modulated by carbon dioxide and/or bicarbonate. With this grant application we plan to test the hypothesis that sAC is the physiological bicarbonate/carbon dioxide sensor in biological systems. Aim 1 will study the direct interactions of bicarbonate with purified recombinant sAC protein using enzyme kinetics, binding assays, and limited proteolysis studies. Aim 2 will study whether bicarbonate activated sAC activity is present in bicarbonate/carbon dioxide regulated physiological systems. Aim 3 will determine with the help of sAC knockout studies in mice whether SAC activity is essential for bicarbonate/carbon dioxide regulated physiological systems in vivo.